miR-146a-Enriched Mesenchymal Stem Cell-Derived Extracellular Vesicles Prolong Corneal Allograft Survival by Modulating Local and Systemic Immune Responses

Abstract

Background: Corneal allograft rejection remains a leading cause of graft failure. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) may provide new options for modulating the immune response to allografts. Objectives: This study aimed to determine whether miR-146a-enriched EVs could prolong corneal allograft survival by modulating local and systemic immune responses. Methods: After corneal allografting from C57BL/6 to BALB/c mice, recipients received subconjunctival injections of PBS, miR-NC-EVs, or miR-146a-EVs isolated from human umbilical cord mesenchymal stem cells (hUCSCs). Local inflammatory and regulatory factors in ocular tissues were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) to quantify the expression of interleukin-1 receptor-associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6), tumor necrosis factor (TNF)-α, and forkhead box protein 3 (Foxp3). Systemic cytokine profiles were assessed in recipient sera using enzyme-linked immunosorbent assay (ELISA) to measure interferon (IFN)-γ, interleukin (IL)-17, IL-10, and transforming growth factor (TGF)-β. Results: Clinical rejection scores were markedly reduced, with significant improvements in opacity, edema, and neovascularization. RT-PCR analysis of corneal tissue showed miR-146a overexpression, downregulation of IRAK1 and TRAF6, a significant reduction in TNF-α mRNA, and increased Foxp3 expression. ELISA analysis of systemic IFN-γ and IL-17 showed that treatment with miR-146a-EVs resulted in lower systemic IFN-γ and IL-17 levels and significantly higher levels of the anti-inflammatory cytokines, IL-10 and TGF-β. Conclusions: This study identifies miR-146a-EVs as a novel cell-free therapy that effectively prolongs corneal transplant survival. The treatment exerts dual immunomodulatory effects by regulating local inflammatory pathways and shifting systemic cytokine profiles toward an anti-inflammatory state, thereby offering a targeted strategy to prevent transplant rejection.